Although all beta-4Gal-T-family members, that are responsible for the synthesis of beta-linked Gal moieties in different oligosaccharides, transfer Gal to GlcNAc, each recognizes differently the remaining monosaccharide units of the oligosaccharide to which GlcNAc is attached. The sequence comparison of the human b4Gal-T family members and the structural homology models based on the 3D structure of b4Gal-T1 reveals only a little or no variation in the GlcNAc binding site among the family members, where as the extended oligosaccharide binding region shows significant variations. This indicates that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site we have carried out the crystal structure analysis of the b4Gal-T1-oligosaccharide complexes, enzyme kinetic analysis and MD simulations. Defining the oligosaccharide binding site of b4Gal-T1 by crystal structure investigations of the complexes with the oligosaccharides : By molecular modeling and docking studies we have previously defined the oligosaccharide binding site of b4Gal-T1, the 3D-structure of which has been determined in our laboratory, either in complex with UDP-galactose and Mn2+ ion, or in complex with alpha-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009304), or of the mutant Met344His-b4Gal-T1 in complex with chitobiose (see Project # Z01 BC 009305). Examination of the GlcNAc binding site in b4Gal-T1 from the crystal structure reveals an "open canal shaped" extended sugar binding site that lay behind the GlcNAc binding site. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. In the crystal structure of b4Gal-T1-LA-complex, LA binds to this region and therefore LA is expected to compete with the GlcNAc containing oligosaccharides, such as chitobiose. Crystallization of the wild type b4Gal-T1with the acceptor either in the presence or absence of UDP has not been successful. This is mainly due to the absence of the acceptor binding-site in the apo-b4Gal-T1 that exists in the open conformation. The enzyme has been crystallized in the closed conformation, where the acceptor site is present, only when UDP-Gal is bound. Although UDP or the acceptor molecules can induce the essential conformational changes, such complexes have been crystallized thus far only in the presence of LA. Since LA binds to the extended sugar binding site it is not possible to crystallize b4Gal-T1 with the oligosaccharide acceptors in the presence of LA. In a previous study we showed (see Project # Z01 BC 009305) that when residue Met344 in bovine b4Gal-T1 is mutated to histidine, the mutant M344H in the presence of Mn2+ and UDP-hexanolamine readily changes to the closed conformation that creates the acceptor binding site, thereby facilitating the structural analysis of the enzyme with various oligosaccharide acceptors. Oligosaccharide substrate interactions with beta-1,4-Galactosyltransferase family members : For a better understanding of the branch specificity of b4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human b4Gal-T1 (h-b4Gal-T1) and the mutant Met340His-b4Gal-T1 (h-M340H-b4Gal-T1) in complex with a GlcNAc containing pentasaccharide and several GlcNAc containing trisaccharides present in N-glycans. These results, together with conformational analysis of oligosaccharides, reveal that, compared to other trisaccharides, the 1,2-1,6-arm trisaccharide, due to its greater conformational flexibility, makes the maximum number of interactions with the protein atoms of h-b4Gal-T1. This correlates with its Km, which is 10-fold lower than that of the 1,2-1,3-arm and 1,4-1,3-arm trisaccharides, and 22-fold lower than that of chitotriose.